High level expression of human cyclooxygenase-2

ABSTRACT

The invention discloses a cDNA consisting of human cyclooxygenase-2 cDNA attached to 3′ flanking sequence of human cyclooxygenase-1 methods for increasing the expression of human cyclooxygenase-2 in transformed cells and assays for preferentially and independently measuring cyclogenase-2 in samples.

This application is a continuation of U.S. patent application Ser. No.08/930,589, filed Feb. 22, 1996, now U.S. Pat. No. 6,107,087, which isthe U.S. national phase of International Patent ApplicationPCT/CA94/00501, filed Sep. 13, 1994, which is a continuation of U.S.patent application Ser. No. 08/084,033, filed Sep. 27, 1993, nowabandoned, and is a continuation-in-part of U.S. patent application Ser.No. 08/064,271, filed May 6, 1993, now U.S. Pat. No. 5,543,297, which isa continuation-in-part of U.S. patent application Ser. No. 07/994,760,filed Dec. 22, 1992, now abandoned, the disclosures of which areincorporated herein by reference, in their entirety.

BACKGROUND OF THE INVENTION

The invention encompasses a system for high level expression of humancyclooxygenase-2 protein including a high expression humancyclooxygenase-2 (COX-2) cDNA and mammalian expression vectors.

Non-steroidal, antiinflammatory drugs exert most of theirantiinflammatory, analgesic and antipyretic activity and inhibithormone-induced uterine contractions and certain types of cancer growththrough inhibition of prostaglandin G/H synthase, also known ascyclooxygenase. Until recently, only one form of cyclooxygenase had beencharacterized, this corresponding to cyclooxygenase-1 (COX-1), aconstitutive enzyme originally identified in bovine seminal vesicles.More recently the gene for an inducible form of cyclooxygenase(cyclooxygenase-2; COX-2)) has been cloned, sequenced and characterizedfrom chicken, murine and human sources. Cyclooxygnase-2 is distinct fromthe cyclooxygenase-1 which has also been cloned, sequenced andcharacterized from sheep, murine and human sources. Cyclooxygenase-2 israpidly and readily inducible by a number of agents including mitogens,endotoxin, hormones, cytokines and growth factors. Given thatprostaglandins have both physiological and pathological roles, we haveconcluded that the constitutive enzyme, cyclooxygenase-1, is responsiblefor much of the endogenous basal release of prostaglandins and hence isimportant in their physiological functions which include the maintenanceof gastrointestinal integrity and renal blood flow. In contrast theinducible form of the enzyme, cyclooxygenase-2, is mainly responsiblefor the pathological effects of prostaglandins where rapid induction ofthe enzyme would occur in response to such agents as inflammatoryagents, hormones, growth factors, and cytokines. Thus, a selectiveinhibitor of cyclooxygenase-2 will have similar antiinflammatory,antipyretic and analgesic properties of a conventional non-steroidalantiinflammatory drug (NSAID), will inhibit hormone-induced uterinecontractions and will have potential anti-cancer effects, but will alsohave a diminished ability to induce some of the mechanism-based sideeffects. In particular, such a selective inhibitor should have a reducedpotential for gastrointestinal toxicity, a reduced potential for renalside effects, a reduced effect on bleeding times and possibly a reducedability to induce asthma attacks in aspirin-sensitive asthmaticsubjects.

Accordingly, it is an object of this invention to provide assays andmaterials to identify and evaluate pharmacological agents that arepotent inhibitors of cyclooxygenase-2 and cyclooxygenase-2 activity.

It is also an object of this invention to provide assays and materialsto identify and evaluate pharmacological agents that preferentially orselectively inhibit cyclooxygenase-2 and cyclooxygenase-2 activity overcyclooxygenase-1 and cyclooxygenase-1 activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C Full length amino acid sequence of a human cyclooxygenase-2protein (SEQ.ID.NO:18).

FIGS. 2A-2C Full length nucleotide sequence of a cloned humancyclooxygenase-2 complementary DNA obtained from human osteosarcomacells (SEQ.ID.NO:19).

FIGS. 3. 749 base flanking sequence for human cyclooxygenase-1complementary DNA obtained from plasmid pcDNA-1-hCOX-1 (SEQ.ID.NO.13).

SUMMARY OF THE INVENTION

The invention encompasses a system for high level expression of humancyclooxygenase-2 protein including a high expression humancyclooxygenase-2 (COX-2) cDNA and mammalian expression vectors.

The invention also encompasses assays to identify and evaluatepharmacological agents that are potent inhibitors of cyclooxygenase-2and cyclooxygenase-2 activity. The invention further encompasses assaysto identify and evaluate pharmacological agents that preferentially orselectively inhibit cyclooxygenase-2 and cyclooxygenase-2 activity overcyclooxygenase-1 and cyclooxygenase-1 activity.

The invention also encompasses recombinant DNA molecules wherein thenucleotide sequence encoding human cyclooxygenase-2 protein is attachedto the 3′ flanking sequence of the gene encoding human cyclooxygenase-1,expression vectors containing COX-2/COX-1 nucleotide sequence fusionsand systems for enhanced expression of human cyclooxygenase-2 protein.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment the invention encompasses an assay for determining thecyclooxygenase-2 activity of a sample comprising the steps of:

(a) adding

(1) a human osteosarcoma cell preparation,

(2) a sample, the sample comprising a putative cyclooxygenase-2inhibitor, and

(3) arachidonic acid; and

(b) determining the amount of prostaglandin E₂ produced in step (a).

For purposes of this specification human osteosarcoma cells are intendedto include, but are not limited to commercially available humanosteosarcoma cell lines, such as those available from the American TypeCulture Collection (ATCC) Rockville, Md. such as osteosarcoma 143B (ATTCCRL 8303) and osteosarcoma 143B PML BK TK (ATCC CRL 8304). We have foundosteosarcoma 143.98.2, which was originally obtained from Dr. WilliamSugden, McArdle Laboratory for Cancer Research, University ofWisconsin-Madlison, to be useful. We have now made a Budapest Treatydeposit of osteosarcoma 143.98.2 with ATCC on Dec. 22, 1992 under theidentification Human osteosarcoma 143.98.2 (now ATCC CRL 11226).

For purposes of this specification the osteosarcoma cell preparationshall be defined as an aqueous monolayer or suspension of humanosteosarcoma cells, a portion of which will catalyze the synthesis ofprostaglandin E₂ (PGE₂). Furthermore the preparation contains a buffersuch as HANK'S balanced salt solution.

Within this embodiment is the genus where the human osteosarcoma cellsare from the osteosarcoma 143 family of cell types which includeosteosarcoma 143B and 143B PML BK TK. We have used osteosarcoma143.98.2.

For purposes of this specification the osteosarcoma cell preparationalso includes human osteosarcoma microsomes, a portion of which willcatalyze the synthesis of PGE₂. The microsomes may be obtained asdescribed below from any of the osteosarcoma cell lines hereindisclosed.

A second embodiment the invention encompasses a composition comprising

(a) an osteosarcoma cell preparation, having between approximately 10³and approximately 10⁹ osteosarcoma cells per ml of cell preparation, and

(b) 0.1 to 50 μl of peroxide-free arachidonic acid per mL of cellpreparation.

Typically the cell preparation will be grown as a monolayer and used inan aliquot of 8.2×10⁴ to 2×10⁶ cells per well (of approximately 1 mLworking volume) as described in the protocol below. Arachidonic acid istypically used in amounts of 1 to 20 μl per well of approximately 1 mLworking volume.

When osteosarcoma microsomes are used instead of whole cells, the cellpreparation will typically comprise 50 to 500 μg of microsomal proteinper mnL of cell preparation. Arachidonic acid is typically used inamounts of 1 to 20 μl acid per mnL of cell preparation.

A third embodiment the invention encompasses an assay for determiningthe cyclooxygenase-1 activity of a sample comprising the steps of:

(a) adding

(1) a cell preparation, the cells expressing cyclooxygenase-1 but notexpressing cyclooxygenase-2,

(2) a sample, the sample comprising a putative cyclooxygenase-1inhibitor,

(3) arachidonic acid; and

(b) determining the amount of PGE₂ produced in step (a).

For purposes of tllis specification cells capable of expressingcyclooxygenase-l but incapable of expressing cyclooxygenase-2, includehuman histiocytic lymphoma cells such as U-937 (ATCC CRL 1593). Suchcells are hereinafter described as COX-1 cells.

For purposes of this specification the cell preparation shall be definedas an aqueous suspension of cells, typically at a concentration of 8×10⁵to 1×10⁷ cells/ml. The suspension will contain a buffer as definedabove.

A fourth embodiment of the invention encompasses a humancyclooxygenase-2 which is shown in FIG. 1 This cyclooxygenase-2 is alsoidentified as SEQ.ID.NO:18. At page 5, lines 24-27, replace the existingparagraph with the following:

A fifth embodiment of the invention encompasses a human cyclooxygenase-2(COX-2) cDNA which is shown in FIG. 2 or a degenerate variation thereof.This cyclooxygenase-2 cDnNA is also identified as SEQ.ID.NO:19.

Within this embodiment is the reading frame portion of the sequenceshown in FIG. 2 encoding the cyclooxygenase-2 shown in FIG. 1; theportion being bases 97 through 1909.

As will be appreciated by those of skill in the art, there is asubstantial amount of redundancy in the codons which are translated tospecific amino acids. Accordingly, the invention also includesalternative base sequences wherein a codon (or codons) are replaced withanother codon, such that the amino acid sequence translated by the DNAsequence remains unchanged. For purposes of this specification, asequence bearing one or more such replaced codons will be defined as adegenerate variation. Also included are mutations (changes of individualamino acids) which produce no significant effect in the expressedprotein.

A sixth embodiment of the invention encompasses a system for stableexpression of cyclooxygenase-2 as shown in FIG. 2 or a degeneratevariation thereof comprising:

(a) an expression vector such as vaccinia expression vector pTM 1,baculovirus expression vector pJVETLZ, pUL941 and pAcmP1 INVITROGENvectors pCEP4 and pcDNAI; and

(b) a base sequence encoding human cyclooxygenase-2 as shown in FIG. 2or a degenerate variation thereof.

In one genus of this embodiment cyclooxygenase-2 is expressed in Sf9 orSf21 cells (INVITROGEN).

A seventh embodiment of the invention encompasses a humancyclooxygenase-2 cDNA useful for high level expression ofcyclooxygenase-2.

Within this embodiment the invention comprises the humancyclooxygenase-2 protein-coding open reading frame cDNA sequence, bases97 to 1909 in FIG. 2, and the human cyclooxygenase-1 flanking region,bases 1 to 749 of FIG. 3, the flanking region being attached to the 3′end of the human cyclooxygensase-2 cDNA.

An eighth embodiment the invention encompasses a system for enhancedstable expression of human cyclooxygenase-2 comprising: (a) anexpression vector such as vaccinia expression vector pTM1, baculovirusexpression vector pJVETLZ, pUL941 and pAcmP1 INVITROGEN vectors pCEP4and pcDNAI; and (b) a base sequence encoding human cyclooxygenase-2 asshown in FIG. 2 or a degenerate variation thereof.

In one genus of this embodiment cyclooxygenase-2 is expressed in Sf9 orSf21 cells (INVITROGEN).

A variety of mammalian expression vectors may be used to expressrecombinant cyclooxygenase-2 in mammalian cells. Commercially availablemammalian expression vectors which may be suitable for recombinantcyclooxygenase-2 expression include but are not limited to pMClneo(Stratagene), pXTI (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC37593), pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224),pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146),pUCTag (ATCC 37460), and gZD35 (ATCC 37565).

DNA encoding cyclooxygenase-2 may also be cloned into an expressionvector for expression in a recombinant host cell. Recombinant host cellsmay be prokaryotic or eukaryotic and include but are not limited tobacteria, yeast, mammalian cells including but not limited to cell linesof human, bovine, porcine, monkey and rodent origin, and insect cellsincluding but not limited to drosophila derived cell lines. Cell linesderived from mammalian species which may be suitable and which arecommercially available, include but are not limited to, CV-1 (ATCC CCL70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61),3T3 (ATCC CCL 92), NIH3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C1271(ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).

The expression vector may be introduced into host cells via any one of anumber of techniques including but not limited to transformation,transfection, protoplast fusion, and electroporation. The expressionvector-containing cells are clonally propagated and individuallyanalyzed to determine whether they produce cyclooxygenase-2 protein.Identification of cyclooxygenase-2 expressing host cell clones may bedone by several means, including but not limited to immunologicalreactivity with anti-cyclooxygenase-2 antibodies, and the presence ofhost cell-associated cyclooxygenase-2 activity.

Expression of cyclooxygenase-2 DNA may also be performed using in vitroproduced synthetic mRNA. Synthetic mRNA can be efficiently translated invarious cell-free systems, including but not limited to wheat germextracts and reticulocyte extracts. Synthetic mRNA can also beefficiently translated in cell-based systems, including but not limitedto microinjection into oocytes, with microinjection into frog oocytesbeing preferred.

To determine the cyclooxygenase-2 cDNA sequence(s) that yields optimallevels of enzymatic activity and/or cyclooxygenase-2 protein,cyclooxygenase-2 cDNA molecules including but not limited to thefollowing can be constructed: the full-length open reading frame of thecyclooxygenase-2 cDNA (base 97 to base 1909 as shown in FIG. 2). Allconstructs can be designed to contain none, all or portions of the 3′untranslated region of cyclooxygenase-2 cDNA (bases 1910-3387).

Cyclooxygenase-2 activity and levels of expression can be determinedfollowing the introduction, both singly and in combination, of theseconstructs into appropriate host cells. Following determination of thecyclooxygenase-2 cDNA cassette yielding optimal expression in transientassays, this cyclooxygenase-2 cDNA construct is transferred to a varietyof expression vectors, including but not limited to mammalian cells,baculovirus-infected insect cells, bacteria such as Escherichia coli,and yeast such as Saccharomyces cerevisiae.

Mammalian cell transfectants, insect cells and microinjected oocytes areassayed for the levels of cyclooxygenase-2 activity and levels ofcyclooxygenase-2 protein by the following methods.

One method for measuring cyclooxygenase-2 enzymatic activity involvesthe incubation of the cells in the presence of 20 μM arachidonic acidfor 10 minutes and measurement of PGE₂ production by EIA.

A second method involves the direct measurement of cyclooxygenase-2activity in cellular lysates or microsomes prepared from mammalian cellstransfected with cyclooxygenase-2 cDNA or oocytes injected withcyclooxygenase-2 mRNA. This assay is performed by adding arachidonicacid to lysates and measuring the PGE₂ production by EIA.

Levels of cyclooxygenase-2 protein in host cells are quantitated byimmunoaffinity and/or ligand affinity techniques. Cyclooxygenase-2specific affinity beads or cyclooxygenase-2 specific antibodies are usedto isolate ³⁵S-methionine labelled or unlabelled cyclooxygenase-2protein. Labelled cyclooxygenase-2 protein is analyzed by SDS-PAGEand/or Western blotting. Unlabelled cyclooxygenase-2 protein is detectedby Western blotting, ELISA or RIA assays employing cyclooxygenase-2specific antibodies.

Following expression of cyclooxygenase-2 in a recombinant host cell,cyclooxygenase-2 protein may be recovered to provide cyclooxygenase-2 inactive form, capable of participating in the production of PGE₂. Severalcyclooxygenase-2 purification procedures are available and suitable foruse. As described above for purification of cyclooxygenase-2 fromnatural sources, recombinant cyclooxygenase-2 may be purified from celllysates and extracts, by various combinations of, or individualapplication of salt fractionation, ion exchange chromatography, sizeexclusion chromatography, hydroxylapatite adsorption chromatography andhydrophobic interaction chromatography.

In addition, recombinant cyclooxygenase-2 can be separated from othercellular proteins by use of an immunoaffinity column made withmonoclonal or polyclonal antibodies specific for full length nascentcyclooxygenase-2.

Whole Cell Assays

For the cyclooxygenase-2 and cyclooxygenase-1 assays, human osteosarcomacells were cultured and used in aliquots of typically 8×104 to 2×10⁶cells/well. We have found it convenient to culture the cells in 1 ml ofmedia in 24-well multidishes (NUNCLON) until they are confluent. Thenumber of cells per assay may be determined from replicate plates priorto assays, using standard procedures. Prior to the assay, the cells arewashed with a suitable buffer such as Hank's Balanced Salts Solution(HBSS; SIGMA), which is preferably prewarmed to 37° C. Approximately 0.5to 2 ml is added per well.

Prior to assay, the appropriate number of COX-1 cells (10⁵ to 10⁷cells/ml) are removed from cultures and concentrated using a techniquesuch as by centrifugation at 300×g for 10 minutes. The supernatant isdecanted and cells are washed in a suitable buffer. Preferably, cellsare again concentrated, such as by centrifugation at 300×.g. for 10minutes, and resuspended to a fmal cell density of approximately 1.5×10⁶cells/ml, preferably in prewarmed HBSS.

Following incubation of human osteosarcoma cells or COX-1 cells in asuitable buffer, a test compound and/or vehicle samples (such as DMSO)is added, and the resulting composition gently mixed. Preferably theassay is performed in triplicate. Arachidonic acid is then added inproportions as described above. We prefer to incubate the cells forapproximately 5 minutes at 30 to 40° C., prior to the addition of the ofperoxide-free arachidonic acid (CAYMAN) diluted in a suitable buffersuch as HBSS. Control samples should contain ethanol or other vehicleinstead of arachidonic acid. A total reaction incubation time of 5 to 10minutes at to 37° C. has proven satisfactory. For osteosarcoma cells,reactions may be stopped by the addition HCl or other acid, preferablycombined with mixing, or by the rapid removal of media directly fromcell monolayers. For U-937 cells, reactions may be advantageously beperformed in multiwell dishes or microcentrifuge tubes and stopped bythe addition of HCl or another mineral acid. Typically, samples assayedin 24-multidishes are then transferred to microcentrifuge tubes, and allsamples frozen on dry ice. Similarly, samples are typically stored at−20° C. or below prior to analysis of PGE₂ levels.

Quantitation of PGE) Concentrations

Stored osteosarcoma 143 and U-937 samples are thawed, if frozen, andneutralized, if stored in acid. Samples are then preferably mixed, suchas by vortexing, and PGE₂ levels measured using a PGE₂ enzymeimmunoassay, such as is commercially available from CAYMAN. We haveadvantageously conducted the plating, washing and color developmentsteps as an automated sequence using a BIOMEK 1000 (BECKMAN). In thepreferred procedure, following the addition of ELLMANS reagent, colordevelopment is monitored at 415 nm using the BIORAD model 3550microplate reader with MICROPLATE MANAGER/PC DATA ANALYSIS software.Levels of PGE₂ are calculated from the standard curve, and mayoptionally determined using BECKMAN IMMlNOFIT EIARIA analysis software.

In the absence of the addition of exogenous arachidonic acid, levels ofPGE₂ in samples from both human osteosarcoma cells and COX-1 cells areapproximately typically 0.1 to 2.0 ng/10⁶ cells. In the presence ofarachidonic acid, levels of PGE₂ increased to approximately 5 to 10 foldin osteosarcoma cells and 50 to 100 fold in COX-1 cells. For purposes ofthis specification, cellular cyclooxygenase activity in each cell lineis defined as the difference between PGE₂ levels in samples incubated inthe absence or presence of arachidonic acid, with the level of detectionbeing approximately 10 pg/sample. Inhibition of PGE₂ synthesis by testcompounds is calculated between PGE₂ levels in samples incubated in theabsence or presence of arachidonic acid.

Microsomal Cyclooxygenase Assay

Human osteosarcoma cells may be grown and maintained in culture asdescribed above. Approximately 10⁵ to 10⁷ cells are plated in tissueculture plates such as available from NUNCLON and maintained in culturefor 2 to 7 days. Cells may be washed with a suitable buffer suchphosphate buffered saline, JIBS), pH 7.2. Cells are then removed fromthe plate, preferably by scraping into PBS. Samples may then beconcentrated, such as by centrifuging at 400×g for 10 minutes at 4° C.Cell pellets or other concentrates are either stored at a suitablereduced temperature such as −80° C., or processed immediately. Allfurther manipulations of the cells are preferably performed at 0-4° C.Cell pellets or concentrates obtained from two tissue culture plates areresuspended in a standard protective buffer, such as Tris-Cl, pH 7.4,containing 10 mM ethylene diaminetetraacetic acid (EDTA), 1 mMphenylmethylsulfonylfluoride, 2 μg/ml leupeptin, 2 μg/ml aprotinin, and2 μg/ml soybean trypsin inhibitor and blended or homogenized, such as bysonication for three×5 seconds using a 4710 series ultrasonichomogenizer (COLE-PARMER) set at 75% duly cycle, power level 3. Enrichedmicrosomal preparations are then prepared, such as by differentialcentrifugation to yield an enriched microsomal preparation. In ourpreferred procedure, the first step consists of four sequentialcentrifugations of the cell homogenate at 10,000×g for 10 min at 4° C.After each centrifugation at 10,000×g the supernatant is retained andrecentrifuged. Following the fourth centrifugation, the supernatant iscentrifuged at 100,000×g for 60-90 min at 4° C. to pellet the microsomalfraction. The 100,000×g supernatant is discarded, and the 100,000×gmicrosomal pellet is resuspended in a suitable buffer such as 0.1 MTris-Cl, pH 7.4, containing 10 mM EDTA and 0.25 mg/ml delipidized bovineserum albumin (BSA) (COLLABORAITIVE RESEARCH INCORPORATED). Theresulting microsomal suspension is recentrifuged such as at 100,000×gfor 90 min at 4° C. to recover the microsomes. Following thiscentrifugation the microsomal pellet is resuspended in a stabilizingbuffer, such as 0.1 M Tris-Cl, pH 7.4, containing 10 mM EDTA at aprotein concentration of approximately 2-5 mg/ml. Aliquots ofosteosarcoma microsomal preparations may be stored at low temperature,such as at −80° C. and thawed prior to use.

As may be appreciated by those of skill in the art, human or serumalbumin or other albumin, may be used as an alternative to BSA.Applicants have found that while the procedure may be carried out usingstandard BSA or other albumin, delipidized BSA is preferred. By use ofdelipidized BSA, endogenous microsomal arachidonic acid can be reducedby a factor of at least 2, such that the arachidonic acid produced inthe assay constituted at least 90% of the total. As may be appreciatedby those of skill in the art, other lipid adsorbing or sequesteringagents may also be used. For purposes of this specification microsomesfrom which the exogenous arachidonic acid has been reduced by a factorof approximately 2 or more shall be considered to be microsomes that aresubstantially free of exogenous arachidonic acid.

COX-1 cells are grown and maintained in culture as described above,washed in a suitable buffer, such as PBS, and cell pellets orconcentrates stored, preferably at −80° C. Cell pellets or concentratescorresponding to approximately 10⁹to 10¹⁰ cells were resuspended in asuitable buffer, such as 10 ml of 0.1 M Tris-HCl, pH 7.4, and are thenblended or homogenized, such as by sonication for 2×5 seconds and 1×10seconds using a 4710 series ultrasonic homogenizer (COLE-PARMER) set at75% duty cycle, power level 3. The cell homogenate is then concentratedand resuspended. In our preferred procedure the cell homogenate iscentrifuged at 10,000×g for 10 minutes at 4° C. The supernatant fractionis then recentrifuged at 100,000×g for 2 hours at 4° C., and theresulting microsomal pellet resuspended in a suitable buffer, such as0.1 M Tris-HCl, 1 mM EDTA, pH 7.4 to a protein concentration ofapproximately 1 to 10 mg/ml. Aliquots of osteosarcoma microsomalpreparations may be stored at reduced temperature and thawed prior touse.

Assay Procedure

Microsomal preparations from Human osteosarcoma and U937 cells arediluted in buffer, such as 0.1 M Tris-HCl, 10 mM EDTA, pH 7.4, (BufferA) to a protein concentration of 50 to 500 μg/ml. 10 to 50 μl of testcompound or DMSO or other vehicle is added to 2 to 50 μl of buffer A. 50to 500 μl of microsomes suspension is then added, preferably followed bymixing and incubation for 5 minutes at room temperature. Typically,assays are performed in either duplicate or triplicate. Peroxide-freearachidonic acid (CAYMAN) in Buffer A is then added to a finalconcentration of 20 μM arachidonic acid, followed by incubation,preferably at room temperature for 10 to 60 minutes. Control samplescontained ethanol or other vehicle instead of arachidonic acid.Following incubation, the reaction was terminated by addition of HCl orother mineral acid. Prior to analysis of PGE₂ levels, samples wereneutralized. Levels of PGE₂ in samples may be quantitated as describedfor the whole cell cyclooxygenase assay.

Cyclooxygenase activity in the absence of test compounds was determinedas the difference between PGE₂ levels in samples incubated in thepresence of arachidonic acid or ethanol vehicle, and reported as ng ofPGE₂/mg protein. Inhibition of PGE₂ synthesis by test compounds iscalculated between PGE₂ levels in samples incubated in the absence orpresence of arachidonic acid.

EXAMPLE 1

Whole Cell Cyclooxygenase Assays

Human osteosarcoma 143.98.2 cells were cultured in DULBECCOS MODIFIEDEAGLES MEDIUM (SIGMA) containing 3.7 g/l NaHCO₃ (SIGMA), 100 μg/lgentamicin (GIBCO), 25 mM HEPES, pH 7.4 (SIGMA), 100 IU/ml penicillin(FLOW LABS), 100 jg/ml streptomycin (FLOW LABS), 2 mM glutamine (FLOWLABS) and 10% fetal bovine serum (GIBCO). Cells were maintained at 37°C., 6% CO₂ in 150cm² tissue culture flasks (CORNING). For routinesubculturing, media was removed from confluent cultures of cells, whichwere then incubated with 0.25% trypsin/0.1 % EDTA (JRH BIOSCIENCES) andincubated at room temperature for approximately 5 minutes. The trypsinsolution was then aspirated, and cells resuspended in fresh medium anddispensed at a ratio of 1:10 or 1:20 into new flasks.

U-937 cells (ATC° C. CRL 1593) were cultured in 89% RPMI-1640 (SIGMA),10% fetal bovine serum (GIBCO), containing 50 lU/ml penicillin (Flowlabs), 50 μg/ml streptomycin (FLOW LABS) and 2 g/l NaHCO₃ (SIGMA). Cellswere maintained at a density of 0.1-2.0×10⁶/ml in 1 liter spinner flasks(Coming) at 37° C., 6% CO₂. For routine subculturing, cells were dilutedin fresh medium and transferred to fresh flasks.

Assay Protocol

For cyclooxygenase assays, osteosarcoma 143.98.2 cells were cultured in1 ml of media in 24-well multidishes (NUNCLON) until confluent. Thenumber of cells per assay was determined from replicate plates prior toassays, using standard procedures. Immediately prior to cyclooxygenaseassays, media was aspirated from cells, and the cells washed once with 2ml of Hanks balanced salts solution (HBSS; SIGMA) prewarmed to 37° C. 1ml of prewarmed HBSS was then added per well.

Immediately prior to cyclooxygenase assays, the appropriate number ofU-937 cells were removed from spinner cultures and centrifuged at 300×gfor 10 minutes. The supernatant was decanted and cells washed in 50 mlof HBSS prewarmed to 37° C. Cells were again pelleted at 300×g for 10minutes and resuspended in prewarmed HBSS to a final cell density ofapproximately 1.5×10⁶ cells/ml. 1 ml aliquots of cell suspension weretransferred to 1.5 ml microcentrifuge tubes or 24-well multidishes(Nunclon).

Following washing and resuspension of osteosarcoma 143 and U-937 cellsin 1 ml of HBSS, 1 μl of test compounds or DMSO vehicle were added, andsamples gently mixed. All assays were performed in triplicate. Sampleswere then incubated for 5 minutes at 37° C., prior to the addition of 10μl of peroxide-free arachidonic acid (CAYMAN) diluted to 1 μM in HBSS.Control samples contained ethanol vehicle instead of arachidonic acid.Samples were again gently mixed and incubated for a further 10 minutesat 37° C. For osteosarcoma cells, reactions were then stopped by theaddition of 100 μl of 1N HCl, with mixing, or by the rapid removal ofmedia directly from cell monolayers. For U-937 cells, reactions inmultiwell dishes or microcentrifuge tubes were stopped by the additionof 100 μl of 1N HCl, with mixing. Samples assayed in 24-multidishes werethen transferred to microcentrifuge tubes, and all samples were frozenon dry ice. Samples were stored at −20° C. prior to analysis of PGE₂levels.

Ouantitation of PGE₂ Concentrations

Osteosarcoma 143.98.2 and U-937 samples were thawed, and 100 μl of 1NNaOH added to samples to which 1N HCl had been added prior to freezing.Samples were then mixed by vortexing, and PGE₂ levels measured using aPGE₂ enzyme immunoassay (CAYMAN) according to the manufacturersinstructions. The plating, washing and colour development steps of thisprocedure were automated using a BIOMEK 1000 (BECKMAN). Following theaddition of ELLNLkNS reagent, color development was monitored at 415 nmusing the Biorad model 3550 microplate reader with microplate manager/PCdata analysis software. Levels of PGE₂ were calculated from the standardcurve determined using BECKMAN IMMUNOFIT EIA/RIA analysis software.

Results

In the absence of the addition of exogenous arachidonic acid, levels ofPGE₂ in samples from both osteosarcoma 143 cells and U-937 cells weregenerally 2 ng/10⁶ cells. In the presence of arachidonic acid, levels ofPGE₂ in samples from these cell lines increased to approximately 5 to 10fold in osteosarcoma cells and 50 to 100 fold in U-937 cells.

Table 1 show the effects of a series of non-steroidal antiinflammatorycompounds on PGE₂ synthesis by human osteosarcoma 143 cells and U-937cells in response to exogenous arachidonic acid.

TABLE 1 osteosarcoma 143 CONCENTRATION PGE₂ U-937 SAMPLE nM ng/10⁶ cellsPGE₂ -AA — 1.8 0.15 AA, no inhibitor — 8.6 17.7 NS-389 100.0 0.8 18.930.0 1.1 17.7 10.0 3.0 20.4 3.0 2.7 18.3 1.0 3.2 17.7 0.3 8.3 18.3ibuprofen 100,000 2.5 1.1 10,000 5.7 5.5 1,000 5.4 14.3 300 10.8 15.8100 12.8 17.1 10 12.5 16.4

EXAMPLE 2

Microsomal Cyclooxygenase Assay

Osteosarcoma 143.98.2 cells were grown and maintained in culture asdescribed above. 3×10⁶ cells were plated in 245×245×20 mm tissue cultureplates (NUNCLON) and maintained in culture for 5 days. Cells were washedtwice with 100 ml of phosphate buffered saline, pH 7.2, (PBS) and thenscraped from the plate with a sterile rubber scraper into PBS. Sampleswere then centrifuged at 400×g for 10 minutes at 4° C. Cell pellets wereeither stored at −80° C. until use or processed immediately. All furthermanipulations of the cells were performed at 0-4° C. Cell pelletsobtained from two tissue culture plates were resuspended in 5 ml of 0.1M Tris-Cl, pH 7.4, containing 10 mM EDTA, 1 mMphenylmethylsulfonylfluoride, 2 μg/ml leupeptin, 2 μg/ml aprotinin, and2 μg/ml soybean trypsin inhibitor and sonicated for three×5 secondsusing a 4710 series ultrasonic homogenizer (Cole-Parmer) set at 75% dutycycle, power level 3. The cell homogenates were then subjected to adifferential centrifugation protocol to yield an enriched microsomalpreparation. The first step consisted of four sequential centrifugationsof the cell homogenate at 10,000×g for 10 min at 4° C. After eachcentrifugation at 10,000×g the supernatant was retained andrecentrifuged. Following the fourth centrifugation, the supernatant wascentrifuged at 100,000×g for 60-90 min at 4° C. to pellet the microsomalfraction. The 100,000×g supernatant was discarded and the 100,000×gmicrosomal pellet was resuspended in 8 mls of 0.1 M Tris-Cl, pH 7.4,containing 10 mM EDTA and 0.25 mg/ml delipidized bovine serum albumin(COLLABORATIVE RESEARCH INCORPORATED). The resulting microsomalsuspension was recentrifuged at 100,000×g for 90 min at 4° C. to recoverthe microsomes. Following this centrifugation the microsomal pellet wasresuspended in 0.1 M Tris-Cl, pH 7.4, containing 10 mM EDTA at a proteinconcentration of approximately 2-5 mg/ml. 500 t aliquots of osteosarcomamicrosomal preparations were stored at −80° C. and thawed on iceimmediately prior to use.

U-937 cells were grown and maintained in culture as described above,washed in PBS, and cell pellets frozen at −80° C. Cell pelletscorresponding to approximately 4×10⁹ cells were resuspended in 10 ml of0.1 M Tris-HCl, pH 7.4 and sonicated for 2×5 seconds and 1×10 secondsusing a 4710 series ultrasonic homogenizer (COLE-PARMER) set at 75% dutycycle, power level 3. The cell homogenate was then centrifuged at10,000×g for 10 minutes at 4° C. The supernatant fraction was thenrecentrifuged at 100,000×g for 2 hours at 4° C., and the resultingmicrosomal pellet resuspended in 0.1 M Tris-HCl, 1 mM EDTA, pH 7.4 to aprotein concentration of approximately 4 mg/ml. 500 μl aliquots ofosteosarcoma microsomal preparations were stored at −80° C. and thawedon ice immediately prior to use.

Assay Protocol

Microsomal preparations from osteosarcoma 143 and U-937 cells werediluted in 0.1 M Tris-HCl, 10 mM EDTA, pH 7.4, (buffer A) to a proteinconcentration of 100 μg/ml. All subsequent assay steps, including thedilution of stock solutions of test compounds, were automated using theBIC)MEK 100 (BIORAD). 5 μl of test compound or DMSO vehicle was added,with mixing, to 20 μl of buffer A in a 96-well minitube plate (BECKMAN).200 μl of microsomes suspension was then added, followed by mixing andincubation for 5 minutes at room temperature. Assays were performed ineither duplicate or triplicate. 25 μl of peroxide-free arachidonic acid(CAYMAN) in buffer A is then added to a final concentration of 20 μMaracidonic acid, with mixing, followed by incubation at room temperaturefor 40 minutes. Control samples contained ethanol vehicle instead ofarachidonic acid. Following the incubation period, the reaction wasterminated by the addition of 25 μl of IN HCl, with mixing. Prior toanalysis of PGE₂ levels, samples were neutralized by the addition of 25μl of 1 N NaOH. Levels of PGE₂ in samples were quantitated by enzymeimmunoassay (CAYMAN) as described for the whole cell cyclooxygenaseassay. The results are shown in Table II.

TABLE II MICROSOMAL ASSAY RESULTS - SET 1 143.98.2 U-937 DRUG %Inhibition % Inhibition 100 nM DuP-697 92 6 3 uM DuP-697 93 48 100 nMFlufenamic 16 5 3 uM Flufenamic 36 0 100 nM Flosulide 13 0 3 uMFlosulide 57 0 100 nM Zomipirac 45 30 3 uM Zomipirac 66 67 100 nM NS-39845 0 3 uM NS-398 64 0 100 nM Diclofenac 70 49 3 uM Diclofenac 86 58 100nM Sulindac sulfide 19 0 3 uM Sulindac sulfide 33 4 100 nM FK-3311 20 03 uM FK-3311 26 0 100 nM Fluribprofen 55 57 3 uM Fluribprofen 58 89

EXAMPLE 3

Reverse Transcriptase/Polymeirase Chain Reaction

In order to confirm the type of cyclooxygenase mRNA present inosteosarcoma 143.38.2 cells, a reverse transcriptase polymerase chainreaction (RT-PCR) analytical technique was employed. Total RNA wasprepared from osteosarcoma cells harvested 1-2 days after the cultureshad reached confluence. The cell pellet was resuspended in 6 ml of 5 Mguanidine monothiocyanate containing 10 mM EDTA, 50 mM Tris-Cl, pH 7.4,and 8% (w/v) β-mercaptoethanol. The RNA was selectively precipitated byaddition of 42 ml of 4 M LiCl, incubation of the solution for 16 h at 4°C., followed by recovery of the RNA by centrifugation at 10,000×g for 90min at 4° C. The RNA pellet which was obtained was resuspended in 10 mMTris-HCl, pH 7.5, 1 mM EDTA, and 0.1 % SDS at a concentration of 4 μg/mland used directly for quantitation of COX-1 and COX-2 mRNAs by RT-PCR.

The quantitative RT-PCR technique employs pairs of syntheticoligonucleotides which will specifically amplify cDNA fragments fromeither COX-1 or COX-2, or the control mRNA,glyceraldehyde-3-phosphate-dehydrogenase (G3PDH). The syntheticoligonucleotides are described in Maier, Hla, and Maciag (J. Biol. Chem.265: 10805-10808 (1990)); Hla and Maciag (J. Biol. Chem. 266:24059-24063 (1991)); and Hla and Neilson (Proc. Natl. Acad. Sci., (USA)89: 7384-7388 (1992)), and were synthesized according to the followingsequences:

Human COX-1 specific oligonucleotides

5′-TGCCCAGCTCCTGGCCCGCCGCTT-3′ SEQ. ID. NO:1:

5′-GTGCATCAACACAGGCGCCTCTTC-3′ SEQ. ID. NO:2:

Human COX-2 specific oligonucleotides

5′-TTCAAATGAGATTGTGGGAAAATTGCT-3′ SEQ. ID. NO:3:

5′-AGATCATCTCTGCCTGAGTATCTT-3′ SEQ. ID. NO:4:

Human glyceraldehyde-3-phosphate dehydrogenase specific oligonucleotides

5′-CCACCCATGGCAAA1TCCATGGCA-3′ SEQ. ID. NO:5:

5′-TCTAGACGGCAGGTCAGGTCCACC-3′ SEQ. ID. NO:6:

The RT-PCR reactions were carried out using a RT-PCR kit fromCETUS-PERKIN ELMER according to the manufacturers instructions. Briefly,4 μg of osteosarcoma total RNA was reverse transcribed to cDNA usingreverse transcriptase and random hexamers as primers for 10 min at 23°C., 10 min at 42° C., followed by an incubation at 99° C. for 5 min. Theosteosarcoma cDNA sample was split into three equal aliquots which wereamplified by PCR using 10 pmol of specific oligonucleotide pairs foreither COX-1 or COX-2, or G3PDH. The PCR cycling program was 94° C. for1 min, 55° C. for 1 min, and 72° C. for 1 min. After the twentieth,twenty-fifth, and thirtieth cycle an aliquot was removed from thereaction mixture and stopped by the addition of 5 mM EDTA. Controlreactions included RT-PCR reactions which contained no RNA and alsoreactions containing RNA but no reverse transcriptase.

Following RT-PCR the reactions were electrophoresed through a 1.2 %agarose gel using a Tris-sodium acetate-EDTA buffer system at 110 volts.The positions of PCR-generated DNA fragments were determined by firststaining the gel with ethidium bromide. The identity of the amplifiedDNA fragments as COX-1, COX-2, or G3PDH was confirmed by Southernblotting, using standard procedures. Nitrocellulose membranes werehybridized with radiolabelled COX-1, COX-2, or G3PDH-specific probes.Hybridization of the probes was detected by autoradiography and also bydetermining the bound radioactivity by cutting strips of thenitrocellulose which were then counted by liquid scintillation counting.

The RT-PCRISouthem hybridization experiment demonstrated that COX-2 mRNAis easily detected in osteosarcoma cell total RNA. No COX-1 cDNAfragment could be generated by PCR from osteosarcoma cell total RNA,although other mRNA species such as that for G3PDH are detected. Theseresults demonstrate that at the sensitivity level of RT-PCR,osteosarcoma cells express COX-2 mRNA but not COX-1 mRNA.

Western Blot of U-937 and 143.98.2 Cell RNA

We have developed a rabbit polyclonal antipeptide antiserum (designatedMF-169) to a thyroglobulin-conjugate of a peptide corresponding to aminoacids 589-600, inclusive, of human cyclooxygenase-2. This amino acidsequence:

Asp-Asp-Ile-Asn-Pro-Thr-Val-Leu-Leu-Lys-Glu-Arg. (also identified hereinas SEQ. ID. NO:7:) has no similarity to any peptide sequence of humancyclooxygenase-1. At a dilution of 1:150, this antiserum detects byimmunoblot a protein corresponding to the molecular weight ofcyclooxygenase-2 in microsomal preparations from osteosarcoma 143 cells.The immunoblot procedure used for these studies has previously beendescribed (Reid et al., J. Biol. Chem. 265: 19818-19823 (1990)). No bandcorresponding to the molecular weight of cyclooxygenase-2 is observedusing a 1:150 dilution of pre-immune serum from the rabbit used to raiseantiserum. Furthermore, a band corresponding to the molecular weight ofcyclooxygenase-2 is observed by immunoblot in microsomal preparations ofosteosarcoma 143 cells using a 1:1 50 dilution of a commerciallyavailable polyclonal antiserum against cyclooxygenase-2 (CAYMAN). Thisantiserum is reported to not cross-react with cyclooxygenase-1. Theseresults demonstrate that osteosarcoma 143 cells expresscyclooxygenase-2. Furthermore, immunoblot analysis with these antiseraand northern blot analysis using a COX-2-specific probe demonstratedthat levels of cyclooxygenase-2 protein and the corresponding mRNAincrease in osteosarcoma 143 cells as they grow past confluence. Withina 3-hour period, and in the presence of 1% serum, human recombinantIL1-α (10 pg/ml; R and D systems Inc.) human recombinant IL1-β (10pg/ml; R and D systems Inc.), human EGF (15 ng/ml; CALBIOCHEM) andconditioned medium from cells grown beyond confluence also increasedlevels of PGE₂ synthesis by osteosarcoma 143 cells in response toarachidonic acid, relative to cells grown in the absence of thesefactors.

EXAMPLE 4

Identification by Northern Blot Analysis of Cell Lines Expressing Eithercox-1 or cox-2 Exclusively

Northern blot analysis was used to determine that U-937 cells expressonly COX-1 mRNA whereas osteosarcoma 143.98.2 expresses only COX-2 mDNAThis was accomplished by first cloning human Cox-2 cDNA from total RNAof the human 143 osteosarcoma cell line. Total RNA was prepared fromapproximately 1×10⁸143 osteosarcoma cells using 4M guanidiniumisothiocyanate (Maniatis, et al., (1982) Molecular Cloning, Cold SpringHarbor). Oligonucleotide primers corresponding to the 5′ and 3′ ends ofthe published Cox-2 cDNA sequence (Hla and Neilson, (1992) Proc. Natl.Acad. Sci., USA 89, 7384-7388) were prepared and are shown below.

HCOX-1 5′CTGCGATGCTCGCCCGCGCCCTG3′ 5′Primer

HCOX-2 5′CTTCTACAGTTCAGTCGAACGTTC3′ 3′Primer

These primers (also identified hereinunder as SEQ. ID. NO:8: and SEQ.ID. NO:9: respectively) were used in a reverse transcriptase PCRreaction of 143 osteosarcoma total RNA. The reaction contained 1 μg of143 osteosarcoma total RNA, which was first reverse transcribed usingrandom hexamers and reverse transcriptase (Maniatis, et al., (1982)Molecular Cloning, Cold Spring Harbor). The products from this reactionwere then amplified using the HCOX-1 and HCOX-2 primers described aboveand Taq polymerase (Saiki, et al., (1988) Science, 239, 487-488). Theconditions used for the amplification were 94° C. for 30 sec, 55° C. for30 sec and 72° C. for 2 min 15 sec for 30 cycles. The amplified productswere run on a 1% low melt agarose gel and the 1.9 kb DNA fragmentcorresponding to the predicted size of human COX-2 cDNA was excised andrecovered. An aliquot of the recovered COX-2 cDNA was reamplified asdescribed above (no reverse transcriptase reaction), the amplifiedproducts were again run on a 1% low melt agarose gel and recovered.

By standard procedures as taught in Maniatis, et al., (1982) MolecularCloning, Cold Spring Harbor, this 1.9 kb DNA fragment was cloned intothe Eco RV site of pBluescript KS (obtained from STRATAGENE) andtransformed into competent DH5a bacteria (obtained from BRL) andcolonies selected on LB agar/ampicillan overnight. Three clones givingthe correct Pst I and Hinc II restriction digestions for human COX-2cDNA were sequenced completely and verified to be correct. This was thefirst indication that the human 143 osteosacoma cell line expressedCOX-2 mRNA.

Northern Analysis

Total RNA from various cell lines and tissues were prepared using theguanidinium isothiocyanate method as described above (Maniatis, et al.,(1982) Molecular Cloning, Cold Spring Harbor). Poly A+ RNA was preparedusing oligo dT cellulose spin columns (Maniatis, et al., (1982)Molecular Cloning, Cold Spring Harbor). The RNA, 10 μg of total or 5 μgof U937 Poly A+ were electrophoresed on 0.9% agarose 2.2 M formaldehydegels (Maniatis, et al., (1982) Molecular Cloning, Cold Spring Harbor).After electrophoresis the gel was washed 3 times for 10 minutes eachwith distilled water and then two times for 30 minutes each in 10×SSC(1×SSC=0.15 M NaCl and 0.015 m sodium citrate). The RNA was transferredto nitrocellulose using capillary transfer (Maniatis, et al., (1982)Molecular Cloning, Cold Spring Harbor) overnight in 10×SSC. The next daythe filter was baked in a vacuum oven at 80° C. for 1.5 hrs to fix theRNA onto the nitrocellulose. The filter was then equilibriated inpre-hybridization buffer (50% formamide, 6×SSC, 50 mM sodium phosphatebuffer pH 6.5, 10 ×Denhardts solution, 0.2% SDS and 250 μg/ml of shearedand denatured salmon sperm DNA) for approximately 4 hours at 40° C. TheCOX-2 cDNA probe was prepared using ³²p dCTP and random hexamer primingwith T7 DNA, polymerase using a commercial kit (Pharmacia).Hybridization was carried out using the same buffer as forpre-hybridization plus 1-3×10⁶ cpmlml of denatured COX-2 cDNA probe at40° C. overnight. The blots were washed two times in 1×SSC and 0.5% SDSat 50° C. for 30 minutes each, wrapped in Saran Wrap and exposed toKodak XAR film with screen at −70° C. for 1-3 days. The same blots werestripped of COX-2 probe by putting them in boiling water and letting itcool to room temperature. The blot was re-exposed to film to ensure allhybridization signal was removed and then pre-hybridized and hybridizedas described above using human COX-1 cDNA as probe. The human COX-1 cDNAwas obtained from Dr. Colin Funk, Vanderbilt University, however thesequence is known in the art (See Funk, C. D., Ftmk, L. B., Kennedy, M.E., Pong, A. S., and Fitzgerald, G. A. (1991), FASEBJ, 5 pp 2304-2312).

Using this Northern blot procedure applicants have established that thehuman 143 osteosarcoma cell line RNA hybridized only to the Cox-2 probeand not to the Cox-1 probe. The size of the hybridizing band obtainedwith the Cox-2 probe corresponded to the correct size of Cox-2 mRNA(approximately 4 kb) suggesting that 143 osteosarcoma cells only expressCox-2 mRNA and no Cox-1 mRNA. This has been confirmed by RT-PCR asdescribed above. Similarly, the human cell line U937 Poly A+ RNAhybridized only to the Cox-1 probe and not to the Cox-2 probe. Thehybridizing signal corresponded to the correct size for Cox-1 mRNA(approximately 2.8 kb) suggesting that U937 only express Cox-1 MnRNA andnot Cox-2. This was also confirmed by RT-PCR, since no product wasobtained from U937 Poly A+ RNA when Cox-2 primers were used (see above).

EXAMPLE 5

Human Cyclooxygenase-2 cDNA and Assays for Evaluating Cyclooxygenase-2Activity Examples demonstrating expression of the Cox -2 cDNA

Comparison of the Cox-2 cDNA sequence obtained by RT-CR of humanosteosarcoma total RNA to the published sequence (Hia, eilson 1992 Proc.Natl. Acad. Sci. USA, 89, 7384-7388), revealed a ase change in thesecond position of codon 165. In the published sequence codon 165 isGGA, coding for the amino acid glycine, whereas in the osteosarcomaCox-2 cDNA it is GAA coding for the amino acid glutamic acid.

To prove that osteosarcoma Cox-2 cDNA codes for glutanic acid atposition 165 we repeated RT-PCR amplification of osteosarcoma Cox-2mRNA; amplified, cloned and sequenced the region surrounding this basechange from human genomic DNA; and used site directed mutagenesis tochange Cox-2glu165 to Cox-2gly165 and compared there activities aftertransfection into COS-7 cells.

1. RT-PCR of Cox-2 mRNA from Human Osteosarcoma Total RNA.

A 300bp Cox-2 cDNA fragment that includes codon 165 was amplified byRT-PCR using human osteosarcoma 143 total RNA. Two primers:

Hcox-13 5′CCTTCCTTCGAAATGCAATTA3′ SEQ.ID.NO:10

Hcox-14 5′AAACTGATGCGTGAAGTGCTG3′ SEQ.ID.NO:11

were prepared that spanned this region and were used in the PCRreaction. Briefly, cDNA was prepared from 1 μg of osteosarcoma 143 totalRNA, using random priming and reverse transcriptase (Maniatis et al.,1982, Molecular Cloning, Cold Spring Harbor). This cDNA was then used asa template for amplification using the Hcox-13 and Hcox-14 primers andTaq polymerase (Saki, et al., 1988, Science, 238, 487-488). The reactionconditions used were, 94° C. for 30s, 52° C. for 30s and 72° C. for 30s,for 30 cycles. After electrophoresis of the reaction on a 2% low meltagarose gel, the expected 300 bp amplified product was obtained, excisedfrom the gel and recovered from the agarose by melting, phenolextraction and ethanol precipitation. The 300 bp fragment was ligatedinto the TAII cloning vector (Invitrogen) and transformed into E. Coli(INVαF) (Invitrogen). Colonies were obtained and 5 clones were picked atrandom which contained the 300 bp insert and sequenced. The sequence ofcodon 165 for all 5 clones was GAA (glutarmic acid). Since the DNAsequence amplified was only 300 bp and the Taq polymerase has quite highfidelity for amplification of smaller fragments and its the secondamplification reaction in which GAA was obtained for codron 165 confirmsthat Cox-2 geRNA from osteosarcoma has GAA for codon 165.

2. Amplificati on of Cox-2 Codon 165 Region from Genomic DNA.

10 To confirm that the osteosarcoma Cox-2 sequence was not an artefactof the osteosarcoma cell line and that this sequence was present innormal cells, the DNA sequences containing codon 165 was amplified fromhuman genomic DNA prepared from normal blood.

The primers used for the amplification reaction were Hcox-13 andHcox-14. The genoic organization of the human Cox-2 gene has not yetbeen determined. Using mouse Cox-2 gene organization as a model for theexon-intron positioning of the human Cox-2 gene would place primerHcox-13 in exon 3 and Hcox-14 in exon 5. The size of the amplifiedproduct would be around 2000 bp based on the mouse Cox-2 geneorganization. The PCR reaction contained 1 μg of human genomic DNA,Hcox-13 and Hcox-14 primers and Taq polymerase. The reaction conditionsused were 94° C. for 30s, 52° C. for 30s and 72° C. for 45s, for 35cycles. An aliquot of the reaction products was separated on a 1% lowmelt agarose gel. Th ere were however a number of reaction products andto identify the correct fragment, the DNA was transferred to a nylonmembrane by southern blotting and probed with a P-32 labelled humanCox-2 internal oligo.

Hcox-17 5′GAGATTGTGGGAAAATTGCTT3′ SEQ.ID.NO:12

Hybridization was to a 1.4 kb DNA frag ment which was recovered from theremainder of the PCR reaction by electrophoresis on a 0.8% low meltagarose gel as described above. This fragment was ligated into the TAIIcloning vector (Invitrogen) and used to transform bacteria (as describedabove). A clone contailnng this insert was recovered and sequenced. Thesequence at codon 165 was Gwe (glutaonic acid) and this sequence wasfrom the human Cox-2 gene since the coding region was interrupted byintrons. (Th e 3′ splice site of intron 4 in human is the same as themouse). This is very convincing evidence of the existence of a humanCox-2 having glutagic acid at position 165.

3. Cox-2gnlu65 vs Cox-2gly65 Activity in Transfected Cos-7 Cells

To determine if Cox-2glul 65 has cyclooxygenase activity and to compareits activity to Cox-2gly165, both cDNA sequences were cloned into theeukaryotic expression vector pcDNA-1 (Invitrogen) and itransfected intoCOS-7 cells (see below). Activity was determined 48h after transfectionby incubating the cells with 20 μM arachadonic acid and measuring PGE₂production by EIA (Cayman). The Cox-2gly165 sequence was obtained bysite directed mutagenesis of Cox-2glu165. Briefly, single stranted KS+plasmid (Stratagene) DNA containing were Cox-2glu165 sequence clonedinto the Eco RV site of the multiple cloning region was prepared byadding 1ml of an overnight bacterial culture (XL-1 Blue (Stratagene)containing the COX-2 plasmid) to 100 ml of LB ampicillian (100 μg/ml)and grown at 37° C. for 1 hr. One ml of helper phage, M13K07,(Pharmacia) was then added and the culture incubated for an additional 7hrs. The bacteria were pelleted by centrifugation at 10,000×g for 10min, ¼ volume of 20% PEG, 3.5 M ammonium acetate was added to thesupernatant and the phage precipitated overnight at 4° C. The singlestranded phage were recovered the next day by centrifugation at 17,000×gfor 15 min, after an additional PEG precipitation the single strandedDNA was prepared from the phage by phenol and phenol:chloroformextractions and ethanol precipitation. The single stranded DNAcontaining the Cox-2glu165 sequence was used as template for sitedirected mutagenesis using the T7-GEN in vitro mutagenesis kit from U.S.Biochemical. The single stranded DNA (1.6 pmoles) was annealed to thephosphorylated oligo HCox-17 (16 pmoles), which changes codon 165 fromGAA to GGA and the second strand synthesis carried out in the presenceof 5-Methyl-dC plus the other standard deoxynucleoside triphosphates, T7DNA polymerase and T4 DNA ligase. After synthesis the parental strandwas nicked using the restriction endonuclease Msp 1 and then removed byexonuclease ImI digestion. The methylated mutated strand was rescued bytransformation of E. coli mcAB-. Colonies were picked, sequenced and anumber of clones were obtained that now had GGA for codon 165 instead ofGAA. This Cox-2gly165 sequence was released from the bluescript KSvector by an Eco R1-Hind III digestion, recovered and cloned into theeukaryotic expression vector pcDNA-1 (Invitrogen) which had also beendigested with Eco R1-Hind III. The Cox-2glu165 sequence was also clonedinto the pcDNA-1 vector in the exact same manner. The only differencebetween the two plasmids was the single base change in codon 165.

The COX-2 pcDNA-1 plasmids were used to transfect Cos-7 cells using amodified calcium phospate procedure as described by Chen and Okyama(Chen, C. A. and Okyama, H. 1988. Biotechniques, 6, 632-638). Briefly,5×10⁵ Cos-7 cells were plated in a 10 cm culture dish containing 10 mlmedia. The following day one hour before transfection the media waschanged. The plasmid DNA (1-30 μl) was mixed with 0.5 ml of 00.25 MCACl₂ and 0.5 ml of 2×BBS (50 mM N-,N-Bis(2-hydroxethyl)-2-amino-ethanesulfonic acid, 280 mM NaCl, 1.5 mMNa₂HPO₄) and incubated at room temperature for 20 min. The mixture wasthen added dropwise to the cells with swirling of the plate andincubated overnight (15-18 hrs) at 35° C. in a 3% CO₂ incubator. Thenext day the media was removed, the cells washed with PBS, 10 ml offresh media added and the cells incubated for a further 48 hrs at 5%CO₂-37° C.

The cells were transfected with 2.5, 5 or 10 μg of Cox-2glu165/pcDNA-1or Cox-2gly165/PcDNA-1. Two plates were used for each DNA concentrationand as a control the cells were transfected with pcDNA-1 plasmid. After48 hours the medium was removed from the cells, the plates were washed3× with Hank's media and then 2 ml of Hank's media containing 20 μMarachadonic acid was added to the cells. After a 20 min incubation at37° C. the medium was removed from the plate and the amount of PGE₂released into the medium was measured by EIA. The PGE₂ EIA was performedusing a commercially available kit (Caymen) following the manufacturersinstructions. Shown in Table III the amount of PGE₂ released into themedia from COS7 cells transfected with pcDNA-1, COS7 transfected withCox-2glu165/pcDNA-1 and COS7 transfected with Cox-2gly165/pcDNA-1.Depending on the amount of DNA transfected into the COS7 cells,Cox-2glu165 is 1.3 to 2.3 times more active than Cox-2glyl 65.

TABLE III LEVEL OF PGE₂ (PG/ML) RELEASED FROM TRANSFECTED COS-7 CELLSPGE₂ pg/ml Amount of Transfected DNA (μg) 2.5 5.0 10.0 Cos-7 +COX-2glu165/pcDNA1 1120 2090 4020 Cos-7 + COX-2gly165/pcDNA1  850 12801770 Cos-7 or Cos-7 + pcDNA1 (5 μg) < 3.9 pg/ml PGE₂

EXAMPLE 6

Vaccinia Virus-directed High Level Expression of Human COX-2 inMammalian Cells Requires a 3′ Non-protein Coding Flanking Sequence

Human COX-2glu165 cDNA was recombined into vaccinia virus using thevaccinia virus transfer vector pTM1 (Moss, et al., (1990) Nature, 348,91-92). Two recombinant vaccinia viruses containing human COX-2 wereconstructed. The first vaccinia virus construct, termed vv-hCOX2-orf,contained only the open reaching frame cDNA sequence coding for theCOX-2 protein. The second vaccinia virus construct, termedvv-hcox-2-3′fl, contained the COX-2 protein-coding open reading framecDNA sequence and the human COX-1 non-coding 3′flanking region attachedto the 3′ end of the COX-2 coding sequence.

The human COX-1 non-coding 3′ flanking region as the following sequence:(SEQ.ID.NO:13)

1      GGGGC AGGAAAGCAG CATTCTGGAG GGGAGAGCTT TGTGCTTGTC 46 ATTCCAGAGTGCTGAGGCCA GGGCTGATGG TCTTAAATGC TCATTTTCTG 96 GTTTGGCATG GTGAGTGTTGGGGTTGACAT TTAGAACTTT AAGTCTCACC 146 CATTATCTGG AATATTGTGA TTCTGTTTATTCTTCCAGAA TGCTGAACTC 196 CTTGTTAGCC CTTCAGATTG TTAGGAGTGG TTCTCATTTGGTCTGCCAGA 246 ATACTGGGTT CTTAGTTGAC AACCTAGAAT GTCAGATTTC TGGTTGATTT296 GTAACACAGT CATTCTAGGA TGTGGAGCTA CTGATGAAAT CTGCTAGAAA 346GTTAGGGGGT TCTTATTTTG CATTCCAGAA TCTTGACTTT CTGATTGGTG 396 ATTCAAAGTGTTGTGTTCCC TGGCTGATGA TCCAGAACAG TGGCTCGTAT 446 CCCAAATCTG TCAGCATCTGGCTGTCTAGA ATGTGGATTT GATTCATTTT 496 CCTGTTCAGT GAGATATCAT AGAGACGGAGATCCTAAGGT CCAACAAGAA 546 TGCATTCCCT GAATCTGTGC CTGCACTGAG AGGGCAAGGAAGTGGGGTGT 596 TCTTCTTGGG ACCCCCACTA AGACCCTGGT CTGAGGATGT AGAGAGAACA646 GGTGGGCTGT ATTCACGCCA TTGGTTGGAA GCTACCAGAG CTCTATCCCC 696ATCCAGGTCT TGACTCATGG CAGCTGTTTC TCATGAAGCT AATAAAATTC 746 GCCC

The relative abilities of these recombinant vaccinate viruses directCOX-2 expression in infected COS7 cells were determined by assaying COXactivity in microsomes isolated from infected cells. Microsomes wereprepared from cells infected for 24 hours with either vv-hCOX2-orf andvv-hcox-2-3′fl. Low levels of COX-2 expression in a mammalian cell lineoccurred when cells were infected with only the open reading frame ofCOX-2 (vv-hCOX2-orf). Higher levels of COX-2 expression were achievedwhen human COX-1 non-coding 3′ flanking region was attached to the 3′end of the COX-2 open reading frame sequence (vv-hcox-2-3′fl).

The vv-hCOX2-orf construct contained only the COX-2 protein-coding openreading frame cDNA sequence encoded by the base sequence 97 to 1912 asshown in FIG. 2. The ATG methionine start codon of the COX-2 cDNA (bases97-99, FIG. 2) was precisely fused to the ATG methionine start codonprovided by the NcoI restriction site of pTM1 (Moss, et al., supra)using the complementary synthetic oligonucleotides GO148 and GO149.

GO148 is

5′-CATGCTCGCCCGCGCCCTGCTGCTGTGCGCGGTCCTGGCGT-3′ (SEQ.ID.NO:14)

GO149 is

5′-CAGGACCGCGCACAGCAGCAGGGCGCGGGCGAG-3′ (SEQ.ID.NO:15)

The annealed oligonucleotides GO148/GO349 en code a modified NcoI sitespanning an ATG nethionine start codon, the first 14 codons of COX-2FIG. 2; Met-Leu-Ala-Arg-Ala-Leu-Leu-Leu-Cys-Ala-Val-Leu-Ala(SEQ.ID.NO:20)), the first nucleotide of the fifteenth codon of COX-2(Leu), and an HaeII restriction enzyme site encoded by the last fivenucleotides of oligonucleotide GO148. A human COX-2 cDNA fragment (FIG.2, bases 133 to 1912) was prepared by digesting plastd pKS-hCOX-2glu165with the restriction enzymes HaeII and BamHI to yeild a 1.9 kilobasepair DNA fragment encoding amino acids 14 to 604 of human COX-2 (FIG.1). The annealed oligonucleotides GO148/GO149 and the HaeII-BamHI humanCOX-2 cDNA fragment were ligated into the vaccinia transfer vector PTM1between the Ncol and BamHI restriction sites to create plasmidpTM1hCOX2-orf.

The vv-hcox-2-3′fl construct contained the COX-2 protein- coding openreading frame cDNA sequence and the human COX-1 non-coding 3′ flankingregion; the 5′ end of the human COX-1 sequence was attached to the 3′end of the COX-2 coding sequence. The human COX-1 non-coding 3′ flankingregion was prepared by polymerase chain reaction (PCR) amplificationusing the plasmid pcDNA-hCOX-1 as a template (Funk, et al., (1991) FASEBJ., 5, 2304-2312). The oligonucleotides used for amplification of thehuman COX-1 3′ flanking sequence are:

GO156 is

5′-CTAGCTAGCTAGAATTCGGGGCAGGAAAGCAGCATTCT-3′ (SEQ.ID.NO:16)

GO157 is

5′-TCGATCGATCGAGGATCCGGGCGAATTTTATTAGCTTCA-3′ (SEQ.ID.NO:17)

Oligonucleotidc GO156 contains an 11 nucleotide clamp sequence (i.e.,5′-CTAGCTAGCTA-3′ (SEQ.ID.NO:21)), an EcoRI restriction site (i.e.,5′-GAATTC-3′), followed by the first 21 nucleotides (i.e.,5′-GGGGCAGGAAAGCAGCATTCT-3′ (SEQ.ID.NO:22)) after the TGA stop codon inthe human COX-1 cDNA sequence (Funk, et al., (1991) FASEB J., 5,2304-2312; see FIG. 3, bases 1-21). Oligonucleotide GO157 contains a 12nucleotide clamp sequence (i.e., 5′-TCGATCGATCGA-3′ (SEQ.ID.NO:23)), aBamHI restriction site (i.e., 5′-GGATCC-3′) and a 21 nucleotide sequencecomplementary to the last 21 nucleotides of the human COX-1 cDNAsequence (Funk, et al., (1991) FASEB J., 5, 2304-2312; see FIG. 3, bases728-749).

The PCR reaction was carried out using a PCR kit from CETUS-PERKIN ELMERaccording to the manufacturers instructions. Briefly, 1 pg of plasmidpcDNA-hCOX-1 (Funk, et al., (1991) FASEB J., 5, 2304-2312) was used as atemplate for PCR amplification by Taq DNA polymerase in the presence of50 picomoles of oligonucleotides GO156 and GO157. The PCR cyclingprogram was 94° C. for 1 min, 55° C. for 1 min, and 72° C. for 1 min for32 cycles. The 784 base pair fragment generated by the PCR was digestedwith BamHI and EcoRI restriction enzymes to remove the clamp sequences.The resulting 749 base pair DNA fragment was ligated to the 3′ codingend of human COX-2 in the plasmid pTM1hCOX2-orf, which had previouslybeen cleaved with EcoRI and BamHI, to create plasmid pTM1-hCOX2-3′fl.

Recombinant vaccinia virus containing human COX-2 sequences weregenerated according to the standard protocols outlined by Ausubel etal., (Current Protocols in Molecular Biology, 2, pp 16.15.1-16.19.9,Wiley & Sons, New York). Plasmids pTM1-hCOX2-orf and pTM1 -hCOX23′flwere recombined into vaccinia virus at the thymidine kinase locus byhomologous recombination. The homologous recombination was carried outby first infecting approximately 1×10⁶ COS-7 cells with 5×10⁵ plaqueforming units (pfu) of wild type vaccinia virus (Western Reserve strain)for 2 hours at 37° C. Two hours post-infection the cells were thentransfected with 5 to 10 μg of the plasmid DNAs pTM1-hCOX2-orf andpTM1-hCOX2-3′fl. The transfection was carried out using a calciumphosphate mammalian transfection kit according to the manufacturer'sinstructions (STRATAGENE). A calcium phosphate precipitate of theplasmid DNAs pTM1-hCOX2-orf and pTM1-hCOX2-3′fl was prepared by mixing 5to 10 μg of DNA in 450 μl H₂O with 50 μl of 2.5 M CaCl₂ and 500 μl of2×N,N-bis (2-hydroxyethyl)-2-aminoethanesulfconic acid in bufferedsaline. The resulting CaPO₄-DNA precipitate was layered onto thevaccinia virus infected-COS7 cells for 4 hours, removed by aspirationand replaced with fresh medium. The virally-infected andplasmid-transfected COS7 cells were incubated for 2 days at 37° C.,harvested by scraping, and disrupted by sonication. The transfected celllysate was used to select and screen recombinant virus plaques.

Selection and screening of recombinant virus plaques were carried out inHuTK-143B cells (ATCC CRL8303) using thymidine kinase selection.Briefly, dilutions of COS7 cell lysates from cells co-transfected withwild-type vaccinia virus and either plasmid pTM1hCOX2-orf or plasmidpTM1-hCOX2-3′fl were plated onto confluent monolayers of HuTK-143B cellsgrown in the presence of 25 μg/ml of bromodeoxyuridine. Under theseconditions viral plaques result only from vaccinia virus that haveundergone a recombination event at the thymidine kinase locus leading toan inactive thymidine kinase gene product. A fraction of the recombinantviruses will result from homologous recombination of the hCOX-2 cDNAsequences in pTM1-hCOX2orf and pTM1-hCOX2-3′fl into the vaccinia virusthyrmidine kinase locus.

Recombinant vaccinia viruses containing hCOX-2 sequences were isolatedby standard selection and screening procedures as outlined by Ausubel etal., (supra). Briefly, recombinant vaccinia viruses were detected bydot-blot hybridization using radioactively labelled DNA probes generatedfrom human COX-1 and COX-2 DNA sequences.

Following three rounds of plaque purification and amplification, tworecombinant vaccinia viruses (vv-hCOX2-orf and vv-hCOX2-3′fl) weregenerated. Both of these viruses contain the entire human COX-2protein-coding cDNA sequence located downstream from a T7 DNA polymerasepromoter sequence integrated into the thymidine kinase locus of vacciniavirus. The construct vv-hCOX2-3′fl also contains the 3′ noncodingflanking region of human COX-1 appended to the 3′ end of the COX-2 gene.

The ability of vv-hCOX2-orf and vv-hCOX2-3′fl to direct the expressionof COX-2 expression was determined by assaying COX-2 activity in COS7cells that had been infected with vaccinia virus constructs. COS7 cellswere grown and maintained in culture as described above. COS7 cells weregrown to a cell density of approximately 1×10⁷ cells in a 175 cm² tissueculture flask in 40 ml of medium and then infected with vaccinia virusat a multiplicity-of-infection of 10:1. Three different infections werecarried out: (a) a control infection using approximately 10⁸ plaqueforming units (pfu) of the helper virus vT7-3 (Moss, et al., (1990)Nature, 348, 91-92) for each 175 cm² tissue culture flask; (b) a testinfection using approximately 10⁸ pfu of the helper virus vT73 andapproximately 10⁸ pfu of vv-hCOX2-orf for each 175 cm² tissue cultureflask; and (c) a test infection using approximately 10⁸ pfu of thehelper virus vT7-3 and about 10⁸ pfu of vv-hCOX2-3′fl for each 175 cm²tissue culture flask. The infections were carried out at 37° C. for 24hours and were followed by cell harvesting and preparation of microsomesas described above. The COX activity in the microsomal fractions wasassayed by determining the level of de novo PGE₂ synthesis fromarachidonic acid as described above. Briefly, each microsomal assaycontained 25 μg of microsomal protein in a total volume of 200 μl of 100mM Tris-OH, pH 7.4, 10 mM EDTA. The reaction was initiated by theaddition of arachidonic acid to a fmal concentration of 20 μM followedby incubation at 23° C. for 40 min. The levels of PGE₂ in samples werequantitated by enzyme immunoassay (CAYMAN) or radioimmunoassay (NEWENGLAND NUCLEAR) as described above for the whole cell cyclooxygenaseassay. For comparison, the levels of COX-2 activity as determined byPGE₂ synthesis in microsomes prepared from osteosarcoma 143B cells andassayed as described above, is presented in Table IV.

TABLE IV MICROSOMAL ASSAY RESULTS OF COX EXPRESSION IN COS-7 CELLSINFECTED WITH VACCINIA VIRUS CONTAINING HUMAN COX-2 CDNA'SCYCLOOXYGENASE ACTIVITY CELL SOURCE ng PGE₂ synthesized/mg FORmicrosomal protein MICROSOME −arachidonic +arachidonic PREPARATIONVACCINIA VIRUS acid acid Osteosarcoma none 5 20 143 COS-7 vT7-3 0.120.37 COS-7 vT7-3 + vv-hCOX2-orf 2.1 14 COS-7 vT7-3 + vv-hCOX2-3′fl 92.53,300

23 24 base pairs nucleic acid single linear cDNA 1 TGCCCAGCTC CTGGCCCGCCGCTT 24 24 base pairs nucleic acid single linear cDNA 2 GTGCATCAACACAGGCGCCT CTTC 24 27 base pairs nucleic acid single linear cDNA 3TTCAAATGAG ATTGTGGGAA AATTGCT 27 24 base pairs nucleic acid singlelinear cDNA 4 AGATCATCTC TGCCTGAGTA TCTT 24 24 base pairs nucleic acidsingle linear cDNA 5 CCACCCATGG CAAATTCCAT GGCA 24 24 base pairs nucleicacid single linear cDNA 6 TCTAGACGGC AGGTCAGGTC CACC 24 12 amino acidsamino acid single linear peptide 7 Asp Asp Ile Asn Pro Thr Val Leu LeuLys Glu Arg 1 5 10 23 base pairs nucleic acid single linear cDNA 8CTGCGATGCT CGCCCGCGCC CTG 23 24 base pairs nucleic acid single linearcDNA 9 CTTCTACAGT TCAGTCGAAC GTTC 24 21 base pairs nucleic acid singlelinear cDNA 10 CCTTCCTTCG AAATGCAATT A 21 21 base pairs nucleic acidsingle linear cDNA 11 AAACTGATGC GTGAAGTGCT G 21 21 base pairs nucleicacid single linear cDNA 12 GAGATTGTGG GAAAATTGCT T 21 749 base pairsnucleic acid single linear Genomic DNA 13 GGGGCAGGAA AGCAGCATTCTGGAGGGGAG AGCTTTGTGC TTGTCATTCC AGAGTGCTGA 60 GGCCAGGGCT GATGGTCTTAAATGCTCATT TTCTGGTTTG GCATGGTGAG TGTTGGGGTT 120 GACATTTAGA ACTTTAAGTCTCACCCATTA TCTGGAATAT TGTGATTCTG TTTATTCTTC 180 CAGAATGCTG AACTCCTTGTTAGCCCTTCA GATTGTTAGG AGTGGTTCTC ATTTGGTCTG 240 CCAGAATACT GGGTTCTTAGTTGACAACCT AGAATGTCAG ATTTCTGGTT GATTTGTAAC 300 ACAGTCATTC TAGGATGTGGAGCTACTGAT GAAATCTGCT AGAAAGTTAG GGGGTTCTTA 360 TTTTGCATTC CAGAATCTTGACTTTCTGAT TGGTGATTCA AAGTGTTGTG TTCCCTGGCT 420 GATGATCCAG AACAGTGGCTCGTATCCCAA ATCTGTCAGC ATCTGGCTGT CTAGAATGTG 480 GATTTGATTC ATTTTCCTGTTCAGTGAGAT ATCATAGAGA CGGAGATCCT AAGGTCCAAC 540 AAGAATGCAT TCCCTGAATCTGTGCCTGCA CTGAGAGGGC AAGGAAGTGG GGTGTTCTTC 600 TTGGGACCCC CACTAAGACCCTGGTCTGAG GATGTAGAGA GAACAGGTGG GCTGTATTCA 660 CGCCATTGGT TGGAAGCTACCAGAGCTCTA TCCCCATCCA GGTCTTGACT CATGGCAGCT 720 GTTTCTCATG AAGCTAATAAAATTCGCCC 749 41 base pairs nucleic acid single linear cDNA 14CATGCTCGCC CGCGCCCTGC TGCTGTGCGC GGTCCTGGCG C 41 33 base pairs nucleicacid single linear cDNA 15 CAGGACCGCG CACAGCAGCA GGGCGCGGGC GAG 33 38base pairs nucleic acid single linear cDNA 16 CTAGCTAGCT AGAATTCGGGGCAGGAAAGC AGCATTCT 38 39 base pairs nucleic acid single linear cDNA 17TCGATCGATC GAGGATCCGG GCGAATTTTA TTAGCTTCA 39 604 amino acids amino acidsingle linear protein 18 Met Leu Ala Arg Ala Leu Leu Leu Cys Ala Val LeuAla Leu Ser His 1 5 10 15 Thr Ala Asn Pro Cys Cys Ser His Pro Cys GlnAsn Arg Gly Val Cys 20 25 30 Met Ser Val Gly Phe Asp Gln Tyr Lys Cys AspCys Thr Arg Thr Gly 35 40 45 Phe Tyr Gly Glu Asn Cys Ser Thr Pro Glu PheLeu Thr Arg Ile Lys 50 55 60 Leu Phe Leu Lys Pro Thr Pro Asn Thr Val HisTyr Ile Leu Thr His 65 70 75 80 Phe Lys Gly Phe Trp Asn Val Val Asn AsnIle Pro Phe Leu Arg Asn 85 90 95 Ala Ile Met Ser Tyr Val Leu Thr Ser ArgSer His Leu Ile Asp Ser 100 105 110 Pro Pro Thr Tyr Asn Ala Asp Tyr GlyTyr Lys Ser Trp Glu Ala Phe 115 120 125 Ser Asn Leu Ser Tyr Tyr Thr ArgAla Leu Pro Pro Val Pro Asp Asp 130 135 140 Cys Pro Thr Pro Leu Gly ValLys Gly Lys Lys Gln Leu Pro Asp Ser 145 150 155 160 Asn Glu Ile Val GluLys Leu Leu Leu Arg Arg Lys Phe Ile Pro Asp 165 170 175 Pro Gln Gly SerAsn Met Met Phe Ala Phe Phe Ala Gln His Phe Thr 180 185 190 His Gln PhePhe Lys Thr Asp His Lys Arg Gly Pro Ala Phe Thr Asn 195 200 205 Gly LeuGly His Gly Val Asp Leu Asn His Ile Tyr Gly Glu Thr Leu 210 215 220 AlaArg Gln Arg Lys Leu Arg Leu Phe Lys Asp Gly Lys Met Lys Tyr 225 230 235240 Gln Ile Ile Asp Gly Glu Met Tyr Pro Pro Thr Val Lys Asp Thr Gln 245250 255 Ala Glu Met Ile Tyr Pro Pro Gln Val Pro Glu His Leu Arg Phe Ala260 265 270 Val Gly Gln Glu Val Phe Gly Leu Val Pro Gly Leu Met Met TyrAla 275 280 285 Thr Ile Trp Leu Arg Glu His Asn Arg Val Cys Asp Val LeuLys Gln 290 295 300 Glu His Pro Glu Trp Gly Asp Glu Gln Leu Phe Gln ThrSer Arg Leu 305 310 315 320 Ile Leu Ile Gly Glu Thr Ile Lys Ile Val IleGlu Asp Tyr Val Gln 325 330 335 His Leu Ser Gly Tyr His Phe Lys Leu LysPhe Asp Pro Glu Leu Leu 340 345 350 Phe Asn Lys Gln Phe Gln Tyr Gln AsnArg Ile Ala Ala Glu Phe Asn 355 360 365 Thr Leu Tyr His Trp His Pro LeuLeu Pro Asp Thr Phe Gln Ile His 370 375 380 Asp Gln Lys Tyr Asn Tyr GlnGln Phe Ile Tyr Asn Asn Ser Ile Leu 385 390 395 400 Leu Glu His Gly IleThr Gln Phe Val Glu Ser Phe Thr Arg Gln Ile 405 410 415 Ala Gly Arg ValAla Gly Gly Arg Asn Val Pro Pro Ala Val Gln Lys 420 425 430 Val Ser GlnAla Ser Ile Asp Gln Ser Arg Gln Met Lys Tyr Gln Ser 435 440 445 Phe AsnGlu Tyr Arg Lys Arg Phe Met Leu Lys Pro Tyr Glu Ser Phe 450 455 460 GluGlu Leu Thr Gly Glu Lys Glu Met Ser Ala Glu Leu Glu Ala Leu 465 470 475480 Tyr Gly Asp Ile Asp Ala Val Glu Leu Tyr Pro Ala Leu Leu Val Glu 485490 495 Lys Pro Arg Pro Asp Ala Ile Phe Gly Glu Thr Met Val Glu Val Gly500 505 510 Ala Pro Phe Ser Leu Lys Gly Leu Met Gly Asn Val Ile Cys SerPro 515 520 525 Ala Tyr Trp Lys Pro Ser Thr Phe Gly Gly Glu Val Gly PheGln Ile 530 535 540 Ile Asn Thr Ala Ser Ile Gln Ser Leu Ile Cys Asn AsnVal Lys Gly 545 550 555 560 Cys Pro Phe Thr Ser Phe Ser Val Pro Asp ProGlu Leu Ile Lys Thr 565 570 575 Val Thr Ile Asn Ala Ser Ser Ser Arg SerGly Leu Asp Asp Ile Asn 580 585 590 Pro Thr Val Leu Leu Lys Glu Arg SerThr Glu Leu 595 600 3387 base pairs nucleic acid single linear cDNA 19GTCCAGGAAC TCCTCAGCAG CGCCTCCTTC AGCTCCACAG CCAGACGCCC TCAGACAGCA 60AAGCCTACCC CCGCGCCGCG CCCTGCCCGC CGCTCGGATG CTCGCCCGCG CCCTGCTGCT 120GTGCGCGGTC CTGGCGCTCA GCCATACAGC AAATCCTTGC TGTTCCCACC CATGTCAAAA 180CCGAGGTGTA TGTATGAGTG TGGGATTTGA CCAGTATAAG TGCGATTGTA CCCGGACAGG 240ATTCTATGGA GAAAACTGCT CAACACCGGA ATTTTTGACA AGAATAAAAT TATTTCTGAA 300ACCCACTCCA AACACAGTGC ACTACATACT TACCCACTTC AAGGGATTTT GGAACGTTGT 360GAATAACATT CCCTTCCTTC GAAATGCAAT TATGAGTTAT GTCTTGACAT CCAGATCACA 420TTTGATTGAC AGTCCACCAA CTTACAATGC TGACTATGGC TACAAAAGCT GGGAAGCCTT 480CTCTAACCTC TCCTATTATA CTAGAGCCCT TCCTCCTGTG CCTGATGATT GCCCGACTCC 540CTTGGGTGTC AAAGGTAAAA AGCAGCTTCC TGATTCAAAT GAGATTGTGG AAAAATTGCT 600TCTAAGAAGA AAGTTCATCC CTGATCCCCA GGGCTCAAAC ATGATGTTTG CATTCTTTGC 660CCAGCACTTC ACGCATCAGT TTTTCAAGAC AGATCATAAG CGAGGGCCAG CTTTCACCAA 720CGGGCTGGGC CATGGGGTGG ACTTAAATCA TATTTACGGT GAAACTCTGG CTAGACAGCG 780TAAACTGCGC CTTTTCAAGG ATGGAAAAAT GAAATATCAG ATAATTGATG GAGAGATGTA 840TCCTCCCACA GTCAAAGATA CTCAGGCAGA GATGATCTAC CCTCCTCAAG TCCCTGAGCA 900TCTACGGTTT GCTGTGGGGC AGGAGGTCTT TGGTCTGGTG CCTGGTCTGA TGATGTATGC 960CACAATCTGG CTGCGGGAAC ACAACAGAGT ATGCGATGTG CTTAAACAGG AGCATCCTGA 1020ATGGGGTGAT GAGCAGTTGT TCCAGACAAG CAGGCTAATA CTGATAGGAG AGACTATTAA 1080GATTGTGATT GAAGATTATG TGCAACACTT GAGTGGCTAT CACTTCAAAC TGAAATTTGA 1140CCCAGAACTA CTTTTCAACA AACAATTCCA GTACCAAAAT CGTATTGCTG CTGAATTTAA 1200CACCCTCTAT CACTGGCATC CCCTTCTGCC TGACACCTTT CAAATTCATG ACCAGAAATA 1260CAACTATCAA CAGTTTATCT ACAACAACTC TATATTGCTG GAACATGGAA TTACCCAGTT 1320TGTTGAATCA TTCACCAGGC AAATTGCTGG CAGGGTTGCT GGTGGTAGGA ATGTTCCACC 1380CGCAGTACAG AAAGTATCAC AGGCTTCCAT TGACCAGAGC AGGCAGATGA AATACCAGTC 1440TTTTAATGAG TACCGCAAAC GCTTTATGCT GAAGCCCTAT GAATCATTTG AAGAACTTAC 1500AGGAGAAAAG GAAATGTCTG CAGAGTTGGA AGCACTCTAT GGTGACATCG ATGCTGTGGA 1560GCTGTATCCT GCCCTTCTGG TAGAAAAGCC TCGGCCAGAT GCCATCTTTG GTGAAACCAT 1620GGTAGAAGTT GGAGCACCAT TCTCCTTGAA AGGACTTATG GGTAATGTTA TATGTTCTCC 1680TGCCTACTGG AAGCCAAGCA CTTTTGGTGG AGAAGTGGGT TTTCAAATCA TCAACACTGC 1740CTCAATTCAG TCTCTCATCT GCAATAACGT GAAGGGCTGT CCCTTTACTT CATTCAGTGT 1800TCCAGATCCA GAGCTCATTA AAACAGTCAC CATCAATGCA AGTTCTTCCC GCTCCGGACT 1860AGATGATATC AATCCCACAG TACTACTAAA AGAACGTTCG ACTGAACTGT AGAAGTCTAA 1920TGATCATATT TATTTATTTA TATGAACCAT GTCTATTAAT TTAATTATTT AATAATATTT 1980ATATTAAACT CCTTATGTTA CTTAACATCT TCTGTAACAG AAGTCAGTAC TCCTGTTGCG 2040GAGAAAGGAG TCATACTTGT GAAGACTTTT ATGTCACTAC TCTAAAGATT TTGCTGTTGC 2100TGTTAAGTTT GGAAAACAGT TTTTATTCTG TTTTATAAAC CAGAGAGAAA TGAGTTTTGA 2160CGTCTTTTTA CTTGAATTTC AACTTATATT ATAAGAACGA AAGTAAAGAT GTTTGAATAC 2220TTAAACACTA TCACAAGATG GCAAAATGCT GAAAGTTTTT ACACTGTCGA TGTTTCCAAT 2280GCATCTTCCA TGATGCATTA GAAGTAACTA ATGTTTGAAA TTTTAAAGTA CTTTTGGGTA 2340TTTTTCTGTC ATCAAACAAA ACAGGTATCA GTGCATTATT AAATGAATAT TTAAATTAGA 2400CATTACCAGT AATTTCATGT CTACTTTTTA AAATCAGCAA TGAAACAATA ATTTGAAATT 2460TCTAAATTCA TAGGGTAGAA TCACCTGTAA AAGCTTGTTT GATTTCTTAA AGTTATTAAA 2520CTTGTACATA TACCAAAAAG AAGCTGTCTT GGATTTAAAT CTGTAAAATC AGATGAAATT 2580TTACTACAAT TGCTTGTTAA AATATTTTAT AAGTGATGTT CCTTTTTCAC CAAGAGTATA 2640AACCTTTTTA GTGTGACTGT TAAAACTTCC TTTTAAATCA AAATGCCAAA TTTATTAAGG 2700TGGTGGAGCC ACTGCAGTGT TATCTCAAAA TAAGAATATC CTGTTGAGAT ATTCCAGAAT 2760CTGTTTATAT GGCTGGTAAC ATGTAAAAAC CCCATAACCC CGCCAAAAGG GGTCCTACCC 2820TTGAACATAA AGCAATAACC AAAGGAGAAA AGCCCAAATT ATTGGTTCCA AATTTAGGGT 2880TTAAACTTTT TGAAGCAAAC TTTTTTTTAG CCTTGTGCAC TGCAGACCTG GTACTCAGAT 2940TTTGCTATGA GGTTAATGAA GTACCAAGCT GTGCTTGAAT AACGATATGT TTTCTCAGAT 3000TTTCTGTTGT ACAGTTTAAT TTAGCAGTCC ATATCACATT GCAAAAGTAG CAATGACCTC 3060ATAAAATACC TCTTCAAAAT GCTTAAATTC ATTTCACACA TTAATTTTAT CTCAGTCTTG 3120AAGCCAATTC AGTAGGTGCA TTGGAATCAA GCCTGGCTAC CTGCATGCTG TTCCTTTTCT 3180TTTCTTCTTT TAGCCATTTT GCTAAGAGAC ACAGTCTTCT CAAACACTTC GTTTCTCCTA 3240TTTTGTTTTA CTAGTTTTAA GATCAGAGTT CACTTTCTTT GGACTCTGCC TATATTTTCT 3300TACCTGAACT TTTGCAAGTT TTCAGGTAAA CCTCAGCTCA GGACTGCTAT TTAGCTCCTC 3360TTAAGAAGAT TAAAAAAAAA AAAAAAG 3387 13 amino acids amino acid singlelinear peptide 20 Met Leu Ala Arg Ala Leu Leu Leu Cys Ala Val Leu Ala 15 10 11 base pairs nucleic acid single linear cDNA 21 CTAGCTAGCT A 11 21base pairs nucleic acid single linear cDNA 22 GGGGCAGGAA AGCAGCATTC T 2112 base pairs nucleic acid single linear cDNA 23 TCGATCGATC GA 12

What is claimed is:
 1. An isolated DNA fragment consisting ofSEQ.ID.NO:13.